TBK1 Antibody

What is TBK1 and what are its primary functions in cellular signaling?

TBK1, also known as tumor necrosis factor (TNF) receptor-associated factor NF-kB activator (TANK)-binding kinase 1, NF-kB-activating kinase (NAK), or T2K, is a multimeric kinase that serves as a pivotal regulator in multiple cellular processes. It plays critical roles in modulating inflammation and autophagy pathways . Recent research has identified TBK1 as a negative regulator of IgA class switching, where B cell-specific TBK1 ablation in mice resulted in uncontrolled production of IgA and development of nephropathy-like disease symptoms .

TBK1 negatively regulates IgA class switching by attenuating noncanonical NF-κB signaling. This mechanism involves TBK1-mediated phosphorylation and subsequent degradation of the NF-κB-inducing kinase . In B cells, TBK1 deficiency promotes the aberrant induction of IgA+ B cells when stimulated with anti-CD40 and BAFF, highlighting its regulatory role in immune responses .

What is the molecular weight of TBK1 and how does this affect antibody selection?

TBK1 has an observed molecular weight of approximately 84-90 kDa . When selecting antibodies for TBK1 detection, researchers should verify that their antibody recognizes a protein at this expected molecular weight. In western blotting applications, Simple Western analysis has confirmed detection of TBK1 at approximately 90 kDa in lysates from Daudi human Burkitt's lymphoma cells and HeLa human cervical epithelial carcinoma cells .

Understanding the precise molecular weight is crucial for experimental planning, as post-translational modifications might cause slight shifts in migration patterns. For instance, when TBK1 is phosphorylated at S172, researchers should account for potential molecular weight changes in gel migration .

What are the recommended applications and dilutions for TBK1 antibodies?

TBK1 antibodies have been validated for multiple experimental applications with specific recommended dilutions:

These dilutions serve as starting points, and researchers should optimize conditions for their specific experimental systems . It is important to note that antibody performance can vary depending on the cell type and experimental conditions.

Different TBK1 antibodies have been validated for specific applications. For example, catalog #28397-1-AP has been tested for WB, IHC, IF/ICC, IP, and ELISA applications with human samples , while catalog #67211-1-PBS has been validated for WB and ELISA applications with human and rat samples .

What cell and tissue types have been validated for TBK1 antibody applications?

TBK1 antibodies have been validated in several cell and tissue types:

For IHC applications with paraffin-embedded tissues, antigen retrieval can be performed with TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0 . In human prostate cancer tissue, TBK1 has been successfully detected using sheep anti-human TBK1 antibody at 3 μg/mL, with specific staining localized to the cytoplasm in epithelial cells .

How does TBK1 phosphorylation affect its detection and biological function?

TBK1 activation involves phosphorylation at S172, which can be specifically detected using phospho-specific antibodies. Research has shown that TBK1 phosphorylation can be induced during viral infection, as demonstrated in U2OS cells infected with adenovirus .

In a time-course experiment, both total TBK1 and phosphorylated TBK1 (pTBK1) levels were analyzed following infection with different adenovirus variants (WT, TS1, and M1). Western blot analysis revealed dynamic changes in TBK1 phosphorylation over time, with detectable changes occurring as early as 30 minutes post-infection .

For accurate assessment of TBK1 activation, researchers should:

• Include both phospho-specific and total TBK1 antibodies in their experiments

• Use appropriate loading controls (such as actin)

• Normalize phosphorylated TBK1 signal to total TBK1 to account for expression variations

• Include non-infected or non-stimulated controls for baseline measurements

What are the optimal conditions for detecting phosphorylated TBK1 in experimental systems?

For optimal detection of phosphorylated TBK1:

• Cell lysis should be performed rapidly with phosphatase inhibitors to preserve phosphorylation status

• Samples should be kept cold throughout processing

• Fresh samples typically yield better results than frozen samples

• Time points should be carefully selected based on the stimulus; for adenovirus infection, early time points (30 min to 3 hours) have shown detectable changes in TBK1 phosphorylation

• Quantification should be performed by normalizing pTBK1 signal to total TBK1 signal

Research has demonstrated that different stimuli may induce varying patterns of TBK1 phosphorylation. For example, in adenovirus infection studies, the wild-type virus (Ad WT) showed different phosphorylation patterns compared to mutant variants (TS1 and M1) .

How can TBK1 antibodies be utilized to study its role in IgA class switching?

TBK1 has been identified as a pivotal negative regulator of IgA class switching . Researchers studying this function can employ TBK1 antibodies in several approaches:

• B cell-specific knockout models: Comparing TBK1 expression between wild-type and B cell-specific TBK1 knockout mice using western blot analysis can confirm deletion efficiency.

• Signaling pathway analysis: TBK1 negatively regulates noncanonical NF-κB signaling. Researchers can examine this by immunoblotting for nuclear p52 and RelB activation, along with cytoplasmic processing of p100 to p52, in both wild-type and TBK1-deficient B cells following stimulation with anti-CD40 and BAFF .

• Class-switching assessment: Following in vitro class-switching stimulation with anti-CD40, BAFF, TGF-β, or IL-4, researchers can analyze:

  • Surface IgA expression by flow cytometry

  • Secreted IgA by ELISA

  • Expression of alpha germline transcripts (αGLT) and activation-induced cytidine deaminase (Aicda) by RT-PCR

Research has demonstrated that TBK1 deficiency promotes the induction of both αGLT and Aicda by anti-CD40 and BAFF stimulation, with particularly pronounced effects on αGLT induction with BAFF stimulation .

What experimental approaches can be used to investigate TBK1's role in autophagy and membrane damage responses?

TBK1 plays an important role in membrane damage responses, particularly in the context of galectin 8-dependent pathways. To study this function, researchers can use TBK1 antibodies in conjunction with other markers:

• Co-localization studies: Immunofluorescence microscopy using antibodies against TBK1 alongside markers for damaged membranes (e.g., galectin-8) and autophagy (e.g., LC3) can reveal spatial and temporal relationships during cellular responses to membrane damage.

• Knockdown/knockout validation: Western blot analysis can be used to confirm successful reduction of TBK1 levels in knockdown or knockout experiments. For example, researchers have examined TBK1, LC3, and actin levels in cells infected with adenovirus for 45 minutes .

• Phosphorylation dynamics: Time-course experiments examining TBK1 phosphorylation at S172 using phospho-specific antibodies can reveal activation patterns in response to various stimuli, such as viral infection .

• Pathway interaction analysis: Immunoprecipitation with TBK1 antibodies followed by immunoblotting for interaction partners can reveal mechanistic details of TBK1's role in autophagy and membrane damage responses.

What are common issues in TBK1 detection and how can they be addressed?

Several technical challenges may arise when working with TBK1 antibodies:

• Non-specific bands in Western blot:

  • Solution: Optimize antibody dilution (start with 1:500-1:2000 for WB)

  • Use freshly prepared samples and appropriate blocking solutions

  • Consider alternative antibody clones if persistent issues occur

• Weak or absent signal in IHC:

  • Solution: Optimize antigen retrieval methods (try both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Increase antibody concentration (try 1:50 dilution for weak signals)

  • Extend incubation time (overnight at 4°C has been successful)

• High background in IF/ICC:

  • Solution: Increase washing steps

  • Optimize blocking and permeabilization conditions

  • Titrate antibody concentration (1:50-1:500 range)

• Variable results between experiments:

  • Solution: Standardize lysate preparation methods

  • Include appropriate positive controls (e.g., HeLa, HT-1080, or HepG2 cells for WB)

  • Ensure consistent sample handling to preserve phosphorylation status

How should cross-reactivity and species specificity be addressed when selecting TBK1 antibodies?

TBK1 antibodies vary in their species reactivity profiles:

• Validated reactivity: Some antibodies, like catalog #28397-1-AP, have been directly tested and validated in human samples, while others like catalog #67211-1-PBS have confirmed reactivity with both human and rat samples .

• Cited reactivity: Broader reactivity has been reported in publications for certain antibodies, including human, mouse, rat, pig, monkey, chicken, bovine, and hamster samples .

When selecting antibodies for cross-species studies:

• Sequence homology: Check the immunogen sequence used to generate the antibody against sequence homology in your species of interest.

• Validation tests: Perform validation experiments with appropriate positive and negative controls from your species of interest.

• Literature search: Look for published studies that have used the antibody in your species of interest.

• Multiple antibody approach: Consider using multiple antibodies targeting different epitopes to confirm findings, especially in less common research species.

How are TBK1 antibodies being used to investigate its role in cancer biology?

TBK1 antibodies have been employed in cancer research across various contexts:

• Expression profiling: TBK1 has been detected in multiple cancer tissues, including human prostate cancer, stomach cancer, and liver cancer . IHC studies have shown specific cytoplasmic localization in epithelial cells of prostate cancer tissue .

• Signaling pathway analysis: As TBK1 is involved in NF-κB signaling, which plays crucial roles in cancer development and progression, antibodies are being used to investigate aberrant TBK1 activation in different cancer types.

• Potential therapeutic targeting: Understanding TBK1 expression and activation in cancer could inform the development of targeted therapies, with antibodies serving as important tools in preclinical research.

For cancer research applications, researchers should consider:

• Using multiple detection methods (WB, IHC, IF) for comprehensive analysis

• Including appropriate cancer and normal tissue controls

• Correlating TBK1 expression with clinical parameters and other signaling molecules

What recent methodological advances have improved TBK1 detection sensitivity and specificity?

Recent technological developments have enhanced TBK1 detection capabilities:

• Simple Western™ technology: This automated capillary-based immunoassay system has been successfully used to detect TBK1 in Daudi human Burkitt's lymphoma cells and HeLa human cervical epithelial carcinoma cells, offering improved quantification compared to traditional Western blotting .

• Multiplexed detection systems: These allow simultaneous detection of total and phosphorylated TBK1 along with other pathway components, providing more comprehensive pathway analysis from limited samples.

• Improved antibody production technologies: Advanced recombinant antibody technologies have led to more specific TBK1 antibodies with reduced lot-to-lot variability.

• Cell-based assays: Development of reporter cell lines and FRET-based assays has enabled more dynamic studies of TBK1 activation in living cells.